Study Design and Population
This was a prospective, multicentre, observational, non-interventional study (Astellas study no. AST-INF-2012-01) conducted in 27 centres and 9 regions across Spain. The date of first enrolment was 18 March 2013 and the last evaluation was completed on 2 January 2015. An Independent Ethics Committee or an Institutional Review Board reviewed and approved the protocol, protocol amendments and patient information before the start of the study. The “master” ethics committee was the H. Clinico San Carlos committee (a list of all ethics approval bodies that approved the study in the various centres is included in the supplementary material). The study was conducted in accordance with the principles of the 1964 Declaration of Helsinki and its later amendments, Good Clinical Practice guidelines and International Conference on Harmonisation guidelines. Informed consent was provided by all patients, or their legal representative, before the start of the study.
Inclusion criteria were male or female patients aged ≥ 18 years admitted to the ICU and requiring empirical antifungal therapy because of a high risk of invasive candidiasis. Patients meeting at least one of the following criteria were considered at high risk: those with a Candida score ≥ 3 (based on severe sepsis, 2 points; surgery, 1 point; parenteral nutrition, 1 point; Candida at multiple sites of infection, 1 point); patients with sepsis and ≥ 3 risk factors for invasive candidiasis (from severe pancreatitis, extra-renal depuration, broad-spectrum antibiotic treatment for > 7 days, abdominal surgery, ICU stay of > 15 days, parenteral nutrition, central venous catheterisation, Candida at multiple sites of infection, candiduria and Acute Physiology and Chronic Health Evaluation score > 15); or immunocompromised patients with sepsis. Patients with documented candidiasis at enrolment, patients taking antifungals at the time of screening or those with any condition that, in the opinion of the investigator, rendered their participation inadvisable were excluded from enrolment.
Treatment and Response Criteria
This was a non-interventional study. Patients were recruited consecutively and managed and treated according to the physicians’ usual clinical practice. The decision to prescribe antifungal therapy was made prior to (and independently from) the patient’s inclusion in the study. Once the decision had been made to initiate treatment, patients were recruited into the study and the pertinent data and samples were collected at enrolment prior to initiating antifungal treatment, which was then administered according to the physicians’ usual practice.
Any other treatments that the patients could receive were according to the treating physician’s regular clinical practice and each site’s health care protocols.
Investigators were asked to make a clinical assessment of the clinical and microbiological response to empirical antifungal treatment for the suspected invasive candidiasis.
Microbiological analyses are summarised in Fig. 1. A blood sample (≥ 10 ml) for culture and serum PCR was taken by peripheral venepuncture, before the first dose of antifungal therapy. Follow-up blood samples for culture and serum PCR were taken according to the site’s usual clinical practice/guideline recommendations 3 and 7 days after the baseline sample. Blood sample collection and management were standardised across the study centres. Samples for PCR were sent to the National Microbiology Centre (CNM).
Blood cultures were processed within each site’s microbiology department, according to usual hospital practice, with identification of isolated strains. When a Candida spp. was isolated, identification and sensitivity tests were performed within each site, according to a standardised protocol. The original blood samples were then frozen and sent to the Reference Centre (National Microbiology Centre, Hospital Clínico San Carlos) to confirm the initial identification and sensitivity results. At the Reference Centre, Candida isolates were identified, and the sensitivity results determined by the minimum inhibitory concentration to antifungal therapy were confirmed by conventional testing, with the original sample used to confirm identification by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.
The remainder of the blood sample used for the PCR assay was centrifuged at 3000 rpm for 10 min to separate the serum, which was then frozen at − 20 °C. The frozen serum samples were stored at each site’s microbiology department until they were sent to the Reference Centre. All the samples were identified by specifically designed labels, and the sites also had specially designed data sheets for accurate sample matching and identification.
Abdominal fluid samples were obtained from the peritoneal cavity during laparotomy or from peritoneal drainage catheters (placed within 24 h of surgery) and were cultured according to each site’s regular clinical practice and subjected to sensitivity testing. When Candida spp. were identified, isolates were sent to the Reference Centre to confirm the initial identification and sensitivity results by conventional testing.
Multiplex Real-Time PCR Assay
Deoxyribonucleic acid (DNA) for PCR assays was extracted from blood and sera using the QIAamp® DNA Mini kit (Qiagen, Izasa, Madrid, Spain), following the manufacturer’s instructions. DNA was eluted in 50 µl of buffer, and 2.2 µl of DNA extracted from each sample was used for subsequent PCR. Two multiplex PCR assays, developed by Fortun et al. (2014), were used to detect six Candida species common in IC (Candida albicans, C. parapsilosis, C. tropicalis, C. glabrata, C. krusei, and C. guilliermondii); these PCR assays have been described in detail elsewhere . Briefly, Candida spp. were detected using molecular probes with fluorescent dyes (FAM, HEX, ROX, and CYAN 500). Probes and primers were designed based on the ITS1/2 sequences of ribosomal DNA regions of Candida strains belonging to the Spanish National Center of Microbiology collection, using Beacon Designer 5.0 software (Premier Biosoft, Palo Alto, CA, USA). Two multiplex PCRs were performed simultaneously in the LightCycler 480 system (Roche Diagnostics, Mannheim, Germany), one using the LightCycler Probes Master Kit (Roche Diagnostic, Madrid, Spain) to detect C. albicans, C. parapsilosis and C. tropicalis and one using the 2 × Sensimix Probe Kit (Quantace, Ecogen, Madrid, Spain) to detect C. glabrata, C. krusei and C. guilliermondii. Samples were analysed in duplicate and standards were run with each set of samples and negative controls. The following CT values were used to define a positive PCR result: C. albicans (20 fg DNA) 34.15–37.44; C. parapsilosis (20 fg DNA) 31.45–35.39; C. tropicalis (20 fg DNA) 32.34–36.16; C. glabrata (200 fg DNA) 31.10–36.24; C. krusei (20 fg DNA) 34–38 (200 fg DNA) 30.80–33.80; C. guilliermondii (20 fg DNA) 38.40–42.31. The overall result was considered positive if both PCRs for each sample were positive according to the laboratory standard. Sensitivity was determined at the Reference Centre; isolates were identified and the sensitivity results, determined by the minimum inhibitory concentration of Candida isolates to antifungal therapy, were confirmed by conventional testing.
Those who conducted the blood/abdominal fluid tests were blinded to the results of the PCR analyses, and vice versa.
A diagnosis of invasive candidiasis was made when a positive blood culture or positive abdominal fluid sample was obtained from a surgical setting or a peritoneal drainage catheter (placed within 24 h of surgery) or when a positive PCR was recorded in samples from patients whose clinical condition improved with antifungal treatment.
The primary end point was the degree of concordance between the results of the serum PCR and blood culture. Secondary end points were the degree of concordance between the results for serum PCR and a positive abdominal fluid culture and positivity for blood culture or abdominal fluid culture (assessed as a composite measure).
The relationship between the presence of invasive candidiasis and the risk factors that contribute to a patient’s high-risk status (Candida score ≥ 3; sepsis and ≥ 3 risk factors for invasive candidiasis; sepsis and immunosuppression) was also investigated.
Data were collected from all patients at the time of enrolment and, if available, when follow-up blood cultures were performed, on discharge from the ICU and at the end of antifungal treatment.
The sample size was calculated based on the primary end point using estimates of the sensitivity of serum PCR (80%) and blood culture (50%), based on the published literature [35, 37], and the kappa index of concordance between both techniques (0.3) in patients with suspected invasive candidiasis, admitted to ICUs in which empirical treatment was initiated with an antifungal. Taking these estimates into account, a sample size of 179 patients would provide precision of ± 0.1 to estimate concordance between serum PCR and blood culture diagnoses, with 95% confidence intervals (CIs). Using the assumption that 10% of patients would not be eligible, the planned sample size was 199. The sample size calculation was performed using EpiData software, version 3.1 (The EpiData Association, Odense, Denmark).
The analysis population included all patients who fulfilled the entry criteria and were considered evaluable. Concordance between the results of serum PCR and blood culture (primary end point) was assessed by estimating the kappa index. The kappa index, which assesses inter-test reliability, can range from − 1 to + 1, with higher scores indicating greater concordance .
The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) and the respective 95% CIs were calculated for serum PCR relative to blood culture.
Positive and negative odds ratios (ORs) and post-test probabilities [positive post-test probability (PPTP) and negative post-test probability (NPTP)] were also determined.
Concordance between the results of serum PCR versus abdominal fluid culture and versus the composite measure were analysed in the same way as the primary end point. The relationship between the presence of invasive candidiasis and patient risk factors (Candida score ≥ 3; sepsis and ≥ 3 risk factors for invasive candidiasis; sepsis and immune suppression) was analysed using a Chi-squared test. Binary logistic regression models were used to determine which risk factors had the greatest weight, according to the microbiological test used. These models were executed hierarchically and with the stepwise backward method. Data were presented as ORs and 95% CIs.
An adjusted binary logistic regression model (with a bilateral significance level of 0.10, Chi-squared test) was used to evaluate which of the individual risk factors were related to the presence of invasive candidiasis according to blood culture results. All calculations were performed with STATA for Windows, version 12 (http://www.stata.com/).